DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Application
The Response filed November 21, 2025 is acknowledged.
Claims 1, 3-4, 9-10, 13-14, 16, 20, 22-23, 32-36, 41-44 and 46-47 are pending and are being examined on the merits.
Response to Arguments
Applicant’s arguments filed November 21, 2025 have been fully considered.
The following rejections are MAINTAINED:
Prior art rejections
Response to arguments regarding prior art rejections
Applicant argues that the prior art rejections should be withdrawn as to instant claim 1 for several reasons (Remarks, p. 5). First, Applicant discusses the differences between Haj-Ahmad Examples 1 and 6. Applicant argues that Example 6 is the only example where miRNA is analyzed and the lysis was performed in a buffer that does not contain a chaotropic salt. In contrast, Applicant argues that Example 1 focuses solely on total RNA isolation, not miRNA isolation. Consequently, the ordinary artisan would not be motivated to use the methods of Example 1 for miRNA isolation (Remarks, p. 5). Applicant further elaborates that Example 1 mentions that the embodiment purifies both large and small RNA, and thus “provides for a broad range of RNA molecules, with tRNA and 5S rRNA possibly being larger than miRNA” and that “Example 1 does not mention purification of miRNAs, nor does it demonstrate that the embodiments of Example 1 could purify miRNA” (Remarks, p. 6).
The Examiner disagrees. Haj-Ahmad teaches that their method is an improvement on prior art RNA purification methods, in that, in prior art methods, small RNAs (<200 nt, which includes miRNAs) are not recovered. In contrast, Haj-Ahmad teaches that modifying the prior art methods, in part, to use a combined silica plus SiC solid support provides RNA purification that “results in an overall higher yield of nucleic acids of all sizes, including small RNAs” (col. 4, ll. 27-30). Thus, Haj-Ahmad clearly teaches that their methods isolate small RNAs, and has clearly defined “small RNAs” to include miRNAs. Whether the method also purifies a broad range of large and small RNAs including tRNA and 55 rRNA is not dispositive as to whether the method purifies miRNAs. Haj-Ahmad clearly states that their methods do so. Haj-Ahmad also clearly defines “small RNAs” to refer to a genus of RNA molecules, comprising, at least, miRNA, siRNA, tRNA and 5S rRNA (col. 4, ll. 10-14). Thus, although Example 1 does not recite the RNA species of miRNA, it does recite the genus of “small RNAs”, which is defined as comprising the miRNA species. Finally, Applicant asserts that the Example 1 reference to the genus of “small RNAs” should be understood to refer to the species of tRNA and 5S RNA (even though they are not explicitly recited), but to exclude the species of miRNAs because they are not explicitly recited, however, Applicant has not provided any argument or evidence as to why “small RNA” in Example 1 should be construed in such a manner.
Applicant additionally argues that the Tanriverdi reference teaches that RNA isolation methods were unpredictable at the time of filing of the instant application. More specifically, Tanriverdi tested several commercially available kits and its own in-house kit for isolating miRNA, and found that the various kits have broad ranges of RNA yield, significant Cq value differences, and varied cDNA, preamplification and qPCR performances (Remarks, pp. 6-7, citing Tanriverdi, abstract; p. 72, col. 1). Applicant asserts that “[g]iven the unpredictability surrounding miRNA purification in the art, a skilled artisan would not recognize that the embodiments of Example 1 in Haj-Ahmad would purify miRNAs” (Remarks, p. 7).
The Examiner agrees that Tanriverdi teaches that there is some unpredictability across various kits and methods for isolating miRNA, but otherwise disagrees with Applicant’s interpretation of Tanriverdi. Specifically, Tanriverdi teaches that all of the kits/methods that were tested successfully isolate miRNA, but that there is variability in the yield of miRNA isolated among the kits/methods tested. Thus, while some kits/methods, including Haj-Ahmad, may result in higher yields of miRNA compared to the others, they all would be expected to isolate at least some miRNA, even if the yield is low. Stated differently, Tanriverdi teaches that there is unpredictability as to the yield of miRNA isolated, but does not teach that there is unpredictability as to whether miRNA is isolated at all. It also should be noted that instant claim 1 is directed to isolating miRNA, but does not require that the method produce any particular minimum yield of miRNA. Further, as noted above, Applicant argues, in reference to Haj-Ahmad Example 1, that the ordinary artisan would not have known to use a buffer which includes a chaotropic salt in a method of isolating miRNA. However, Tanriverdi teaches using a chaotropic salt (specifically, guanidine-HCl) in their in-house kit/method, which they report as producing the highest yield of miRNA (abstract; p. 67, left col., para. 3). Thus, the use of chaotropic salts in miRNA purification methods was known in the art at the time of filing the instant application.
Finally, Applicant argues that the various additional secondary references cited against the dependent claims do not cure the deficiencies of the rejection as to claim 1 (Remarks, pp. 7-8). As noted, the Examiner disagrees with Applicant’s characterization of the deficiencies of the references cited against claim 1, and thus disagrees with these arguments, as well.
These arguments are not persuasive. The rejections are maintained.
Information Disclosure Statement
The Information Disclosure Statement submitted November 21, 2025 has been considered.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 3-4, 9-10, 14, 16, 20, 22-23, 32, 34-36 and 41 are rejected under 35 U.S.C. 103 as being unpatentable over Haj-Ahmad1 (US Patent No. 9,422,596) in view of Kirsch2 (US Patent App. Pub. No. 2013/0041145), as evidenced by 3Norgen Biotek Corporation (Total RNA Purification Kit, Product Insert, 2022; hereinafter, “Norgen”) and 4Norgen Biotek Corporation (Buffer RL, Safety Data Sheet, 2015; hereinafter, “Norgen SDS”).
Regarding independent claim 1, Haj-Ahmad teaches …
A method for purifying unbiased miRNA from a biological sample consisting of blood, the method comprising: (col. 1, ll. 35-37: “the method of nucleic acid isolation being employed does not favor the isolation of certain sizes of RNA molecules”; col. 3, l. 19: “method can be used to isolate RNA”; col. 4, ll. 21-23: “silicon carbide based purification methods have been shown to exhibit no size-bias when used for RNA purification”; col. 9, ll. 39-41: “isolate nucleic acids from … blood”; col. 15, ll. 23-29: “the purified RNA was used in RT-qPCR reactions for the detection of … microRNA … specific primers for … miR-30b and miR-21”);
(a) obtaining the biological sample, dissolved in a lysis reagent (col. 9, ll. 39-41: “isolate nucleic acids from … blood”);
(b) lysing the biological sample dissolved in the lysis reagent (col. 9, ll. 43-46: “biological samples can be prepared using conventional … lysis methods (as appropriate to the sample type) to provide an aqueous solution containing the nucleic acids to be recovered”; col. 10, ll. 45-46: Lysis Solution from Norgen’s Total RNA Purification Kit (Cat# 17200, Norgen, Thorold Canada);
(c) purifying miRNA from the lysed sample, wherein the purifying does not involve an RNA precipitation step (col. 4, ll. 44-46: “a solid support for the isolation and purification of nucleic acids, the solid support comprising silica and [silicon carbide]”; col. 5, ll. 34-35: “the solid support is provided in a spin column”; col. 9, ll. 53-57 through col. 10, ll. 1-4: “the solid support can comprise a slurry of silica particles and [silicon carbide] particles … slurry mixture can be added to aqueous solution to allow the nucleic acids to bind to the … particles … solid support with the bound nucleic acids can then be separated … the nucleic acids can be eluted … and collected for downstream applications”);
wherein purifying comprises applying the lysed sample to a silica spin column to bind the miRNA to said column (col. 3, ll. 1-3; col. 10, ll. 5-8);
and (d) selectively analyzing the purified miRNA (col. 15, ll. 23-29: “the purified RNA was used in RT-qPCR reactions for the detection of … microRNA … specific primers for … miR-30b and miR-21”);
wherein the purified miRNAs provide a representative unbiased population of the miRNA content of the biological sample (col. 4, ll. 21-30: “silicon carbide based purification methods have been shown to exhibit no size-bias when used for RNA purification … using silica and [silicon carbide] in combination results in an overall higher yield of nucleic acids of all sizes, including small RNAs”; col. 9, ll. 39-41; col. 3, l. 19).
Haj-Ahmad teaches blood as noted above, but does not expressly teach “whole blood”, nor does Haj-Ahmad indicate that the lysis reagent was a “non-precipitating” lysis reagent.
Kirsch teaches that the sample consists of whole blood (abstract). Kirsch also teaches a non-precipitating lysis reagent, and that the lysis reagent comprises a chaotropic salt, specifically guanidium thiocyanate (para. 43: “the aqueous lysis solution is mixed with the sample”; para. 45: “sample can be brought directly in contact with the lysis solution”; para. 47: “lysis substance can … contain a [non-precipitating] chaotropic salt”; see also Example 1, paras. 85-90: describes lysis reagent with GuSCN).
Regarding the teaching in Haj-Ahmad regarding the use of Lysis Solution from Norgen’s Total RNA Purification Kit, it is noted that the Norgen lysis buffer comprises GuSCN (Norgen, p. 1, para. 2: “process involves first lysing the cells … with the provided Bufffer RL”; Norgen SDS, p. 2, section 3: guanidinium thiocyanate). As evidenced by these references, Haj-Ahmad’s method did employ a non-precipitating lysis buffer that contained the same chaotropic salt, guanidium thiocyanate, as Kirsch’s method. Thus, the only difference between the method of claim 1 and Haj-Ahmad’s method is the use of “whole blood”, rather than “blood”.
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to practice the method of Haj-Ahmad using whole blood. Firstly, one of ordinary skill in the art would have assumed Haj-Ahmad’s reference to “blood” meant “whole blood”. Secondly, Kirsch demonstrates that it was known and conventional in the art to isolate nucleic acids from “whole blood”.
The ordinary artisan would also have been motivated to incorporate the Kirsch whole blood sample in the Haj-Ahmad method, as Haj-Ahmad specifically teaches that the method can be used on blood, although does not further qualify the type of blood. It would have been obvious to the ordinary artisan to try the whole blood sample in the Haj-Ahmad method, with an expectation of success as Haj-Ahmad teaches a wide variety of biological samples, including blood, and does not limit the specific types of blood samples that may be used.
Regarding dependent claim 3, Kirsch suggests collecting whole blood in a tube comprising the lysis reagent (para. 17: describes a direct lysis prior art method of collecting blood in a tube comprising lysis reagent; para. 25: teaches that direct processing sample is desirable).
Regarding dependent claim 4, Kirsch teaches inactivating nucleases (para. 32: RNases) and suggests inactivating microbes (para. 15: describes prior art method using GuSCN that inactivates pathogens; Example 1, paras. 85-90: describes lysis reagent with GuSCN). As noted above, Haj-Ahmad used a lysis reagent from the Norgen kit containing GuSCN, and so would have inactivated nucleases and pathogens, as noted by Kirsch.
Regarding dependent claim 9, Kirsch additionally teaches wherein the lysis is performed at 20-30°C (para. 95, step 6: 1 min at room temperature), while Haj-Ahmad additionally suggests wherein the purifying is performed at 20-30°C. That is, Haj-Ahmad teaches that the purifying step is performed, in part, using Norgen’s Lysis, Wash and Elution Solutions (Example 2; col. 11, ll. 33-52). Norgen teaches that each of these solutions is intended to be used at room temperature (p. 5: “all solutions are at room temperature prior to use”).
Regarding dependent claim 10, Kirsch additionally teaches wherein the lysis step involves an incubation of at least 1 minute (para. 95, step 6: 1 min at room temperature).
Regarding dependent claims 14 and 16, Kirsch teaches wherein the lysis reagent and sample are mixed at a volume of 0.7-1.5: 0.7-1.5 lysis reagent: sample (para. 43: 1.5:1 to 1:1.5, 1.2:1 to 1:1.2, as recited in claim 14, and wherein the lysis reagent and sample are mixed at 1:1 vol: vol (para. 43), as recited in claim 16.
Regarding dependent claims 22 and 23, as noted above, Haj-Ahmad’s method employed a lysis reagent with a chaotropic salt (GuSCN). In addition, Kirsch teaches that the lysing step further comprises proteinase K digestion (para. 90), as recited in claim 22, and that the lysing step further comprises agitation with one or more beads (para. 56), as recited in claim 23.
Regarding dependent claim 20, Haj-Ahmad’s method did not employ phenol.
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to practice the method of Haj-Ahmad using whole blood in view of Kirsch, as discussed above, and further to modify the method according to the teachings of Kirsch by employing the dilution ratios of sample to lysis reagent, the times and temperatures of the incubation steps, and the use of beads and proteinase K digestion as taught by Kirsch, as Kirsch taught these modifications as useful for the extraction of nucleic acids from whole blood.
The ordinary artisan would have been further motivated to collect the blood in a tube with the lysis reagent in order to reduce the number of steps and increase the efficiency of the method.
Regarding dependent claim 32, Haj-Ahmad additionally teaches that the lysed sample is diluted in an alcohol, specifically ethanol, prior to applying said lysed sample to the column (col. 10, ll. 47-48). Kirsch also teaches diluting the mixture in alcohol prior to applying the mixture to the column, and teaches that either ethanol or isopropanol are suitable for that purpose (para. 59). Kirsch does not specifically teach that proportion should be an equal volume of lysate to isopropanol, but does give some guidance on appropriate alcohol content of the final mixture. The ordinary artisan would have been able to optimize both the particular alcohol and the appropriate dilution volume through routine optimization to arrive at the claimed limitation.
Regarding dependent claim 34, Haj-Ahmad additionally teaches that the purifying further comprises removal of the guanidium thiocyanate (col. 10, ll. 18-22: “spin column can be washed … to remove materials not bound to the solid support”).
Regarding dependent claim 35, Haj-Ahmad teaches washing the column with a buffer comprising ethanol (col. 10, ll. 52-55: “Wash Solution from Norgen’s Total RNA Purification Kit … was added to each column … wash step was repeated two more times). In addition, Norgen teaches that the Wash Solution is supplied as a concentrated solution to which ethanol is to be added by the end user (p. 5). Thus, the Norgen Wash Solution used on the column comprises ethanol.
Regarding dependent claim 36, Haj-Ahmad’s method does not employ phase separation (i.e. an organic extraction such as the Trizol/BCP method).
Regarding dependent claim 41, Haj-Ahmad additionally teaches selectively analyzing the purified miRNA by performing qRT-PCR (col. 15, ll. 23-29).
Claims 13 and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Haj-Ahmad (US Patent No. 9,422,596) in view of Kirsch (US Patent App. Pub. No. 2013/0041145) as evidenced by Norgen Biotek Corporation (Total RNA Purification Kit, Product Insert, 2022; hereinafter, “Norgen”) and Norgen Biotek Corporation (Buffer RL, Safety Data Sheet, 2015; hereinafter, “Norgen SDS”), as applied to claims 1 and 10 above, and further in view of Schuster (WO 2017/137573 A1; citations below are to English version of application, US Patent App. Pub. No. 2019/01277295).
Regarding dependent claim 13, Schuster additionally teaches wherein the incubation
comprises storing the sample at less than 10°C for at least one day (para. 40: “at least 0°C … up to 22.5°C”).
Regarding dependent claim 33, Kirsch additionally teaches wherein the purifying further comprises performing DNase digestion (paras. 60-61), while Schuster specifically teaches DNase I (para. 61).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to practice the method suggested by the combined teachings of Haj-Ahmad and Kirsch, discussed above, and to store the sample in the lysis reagent prior to further processing. The ordinary artisan would have been motivated to do so with the expected advantage that it would improve the collection and transport of the blood sample to the testing facility. The ordinary artisan would have been further motivated to perform DNase digestion, as taught by Kirsch, to increase the purity of the final product, and to select DNAse I, in particular, as taught by Schuster, as it is obvious to select a known material based on its suitability for its intended use. The ordinary artisan would have had an expectation of success as optimizing nucleic acid isolation methods is well-known in the art.
Claims 42-44 are rejected under 35 U.S.C. 103 as being unpatentable over Haj-Ahmad (US Patent No. 9,422,596) in view of Kirsch (US Patent App. Pub. No. 2013/0041145), as evidenced by Norgen Biotek Corporation (Total RNA Purification Kit, Product Insert, 2022; hereinafter, “Norgen”) and Norgen Biotek Corporation (Buffer RL, Safety Data Sheet, 2015; hereinafter, “Norgen SDS”), as applied to claim 1 above, and further in view of Knutsen6 (Performance Comparison of Digital microRNA Profiling Technologies Applied on Human Breast Cancer Cell Lines, PLOS ONE, 8(10, e75813, 2013), as evidenced by Illumina7 (TruSeq Small RNA Sample Preparation Guide, 2010).
Regarding dependent claims 42-44, Knutsen teaches constructing a library for miRNA
sequencing and performing next generation miRNA sequencing on said library (p. 2, right col., paras. 3-4), as recited in claim 42. In addition, Knutsen as evidenced by Illumina teaches ligating adaptors to each end of the microRNAs, as recited in claim 43, and wherein the adaptors comprise barcodes, as recited in claim 44. Specifically, Knutsen teaches Illumina HiSeq next generation sequencing (p. 2, right col., paras. 3-4), while Illumina teaches ligating adaptors to each end of the microRNAs (Fig. 1), and where the adapters comprise an index sequence (i.e., a barcode) which is incorporated into the adapter during PCR (Fig. 1).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to practice the method suggested by the combined teachings of Haj-Ahmad and Kirsch, discussed above, and to construct a library for and performing next generation sequencing, as taught by Knutsen, as doing so merely represents combining prior art elements according to known methods to yield predictable results (MPEP 2143(I)). The ordinary artisan would have had an expectation of success as preparing libraries and performing nucleic acid sequencing is well-known in the art.
Claims 46-47 are rejected under 35 U.S.C. 103 as being unpatentable over Haj-Ahmad (US Patent No. 9,422,596) in view of Kirsch (US Patent App. Pub. No. 2013/0041145), as evidenced by Norgen Biotek Corporation (Total RNA Purification Kit, Product Insert, 2022; hereinafter, “Norgen”) and Norgen Biotek Corporation (Buffer RL, Safety Data Sheet, 2015; hereinafter, “Norgen SDS”) in view of Knutsen (Performance Comparison of Digital microRNA Profiling Technologies Applied on Human Breast Cancer Cell Lines, PLOS ONE, 8(10, e75813, 2013), as evidenced by Illumina (TruSeq Small RNA Sample Preparation Guide, 2010) as applied to claim 42 above, and further in view of Hsiao8 (Gene Sets Enrichment Analysis of miRNA Expression Profile, 2011 IEEE International Conference on Bioinformatics and Biomedicine Workshops, 12460463, 2011).
Regarding dependent claims 46-47, Hsiao teaches performing unbiased miRNA
functional enrichment analysis (e.g., abstract), as recited in claim 46, and where said analysis comprises using a target prediction program, gene annotation data and applying statistical analysis (p. 450, Analysis Framework), as recited in claim 47.
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to practice the method suggested by the combined teachings of Haj-Ahmad, Kirsch and Knutsen, discussed above, and to perform the particular data analysis of Hsiao, as doing so merely represents combining prior art elements according to known methods to yield predictable results (MPEP 2143(I)). The ordinary artisan would have had an expectation of success as performing data analysis is well-known in the art.
Conclusion
Claims 1, 3-4, 9-10, 13-14, 16, 20, 22-23, 32-36, 41-44 and 46-47 are being examined, and are rejected. No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CAROLYN GREENE whose telephone number is (571)272-3240. The examiner can normally be reached M-Th 7:30-5:30 EST.
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/CAROLYN L GREENE/Primary Examiner, Art Unit 1681
1 Haj-Ahmad was cited in the PTO-892 Notice of References Cited mailed June 24, 2022.
2 Kirsch was cited in the PTO-892 Notice of References Cited mailed November 10, 2021.
3 Norgen was cited in the PTO-892 Notice of References Cited mailed June 24, 2022.
4 Norgen SDS was cited in the PTO-892 Notice of References Cited mailed June 24, 2022.
5 Schuster ‘729 application was cited in the PTO-892 Notice of References Cited mailed November 10, 2021.
6 Knutsen was cited in the PTO-892 Notice of References Cited mailed November 10, 2021.
7 Illumina was cited in the PTO-892 Notice of References Cited mailed June 24, 2022.
8 Hsiao was cited in the PTO-892 Notice of References Cited mailed November 10, 2022.